Antithrombotic agent and humanized anti-von willebrand factor monoclonal antibody

ABSTRACT

Anti-thrombotic agents containing humanized antibodies which bind to von Willebrand factor.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] Humanized monoclonal antibodies against human von Willebrandfactor, cells which produce the antibodies, and antithrombotic agentscontaining the foregoing antibodies as the active ingredient.

[0003] 2. Background of the Invention

[0004] When subendothelium tissue is exposed due to injury, plateletsflowing through the bloodstream immediately adhere to thesubendothelium. This event triggers a series of platelet activationprocesses including platelet aggregation and release of intracellulargranules, after which a thrombus is formed and bleeding stops. Thrombusformation is necessary for the physiological hemostatic mechanism.However, the thrombus can cause a number of thrombotic diseases such asmyocardial infarction, angina pectoris, cerebral infarction and cerebralthrombosis.

[0005] Many anti-thrombotic agents have been developed to treatthrombotic diseases. However, many conventional antithrombotic agentshave low effectiveness in clinical applications and have lowthrombus-specificity, causing hemorrhaging as a side effect.

[0006] An important protein which functions at the early stage ofthrombus formation is von Willebrand factor (“vWF”), in blood plasma.Hemorrhagic legions associated with the occurrence of qualitative andquantitative changes in vWF are indications of von Willebrand disease(“vWD”). Several antibodies against vWF are known: NMC-4 disclosed byFujimura et al, J. Nara Med. Assoc., vol. 36, 662 (1985); RFF-VIIIRAG:1disclosed by Tuddenham et al, Blood, vol. 177, no. 1, 113 (1992); andthe monoclonal antibodies produced by hybridomas AJvW-1, AJvW-2, AJvW-3,and AJvW-4 disclosed by Nagano et al, PCT/JP95/02435 (incorporatedherein by reference).

[0007] The present invention provides humanized antibodies based on theantibodies produced by hybridoma AJvW-2. This murine monoclonal antibodyis an effective inhibitor of the physiological activity of vWF and wouldbe desirable to use for treating thrombotic diseases. Unfortunately, theuse of murine monoclonal antibodies such as those from AJvW-2 havecertain drawbacks in human treatment, particularly in repeatedtherapeutic regimens. And mouse monoclonal antibodies tend to have ashort half-life in humans and lack other important immunoglobulinfunctional characteristics when used in humans. More importantly, murinemonoclonal antibodies contain substantial amino acid sequences that areimmunogenic when injected into human patients. Numerous studies haveshown that, after injection of foreign antibodies, the immune responseelicited in a patient against the injected antibody can be quite strong,eliminating the antibody's therapeutic effectiveness after the initialtreatment. Moreover, if mouse or other antigenic (to humans) monoclonalantibodies are used to treat a human disease, then subsequent treatmentswith unrelated mouse antibodies may be ineffective or even dangerous dueto cross-reactivity.

[0008] While the production of so-called “chimeric antibodies” (e.g.,mouse variable regions joined to human constant regions) has provensomewhat successful, significant immunogenicity problems remain. (See,LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. USA, 86, 4220-4224(1989); M. N. Saleh et al., Human Antibod. Hybridomas e: 19 (1992)).

[0009] In general, producing human immunoglobulins reactive with vonWillebrand factor with high affinity would be extremely difficult usingtypical human monoclonal antibody production techniques. Thus, there isa need for improved forms of humanized immunoglobulins specific for vonWillebrand factor that are substantially non-immunogenic in humans, yeteasily and economically produced in a manner suitable for therapeuticformulation and other uses. The present invention fulfills these andother needs.

SUMMARY OF THE INVENTION

[0010] An object of the invention is to provide humanizedimmunoglobulins, such as monoclonal antibodies, against von Willebrandfactor, humanized forms of mouse antibody AJvW-2, polynucleotidesequences encoding the immunoglobulins; a method for producing theimmunoglobulins; pharmaceutical compositions comprising theimmunoglobulins as an active ingredient; a therapeutic agent fortreating thrombotic diseases comprising the antibody as an activeingredient; and a method for treating such diseases.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1(a) is the heavy chain variable region sequence of AJvW-2,SEQ ID NO:1.

[0012]FIG. 1(b) is the light chain variable region sequence of AJvW-2,SEQ ID NO:2.

[0013]FIG. 2(a) is the heavy chain variable region sequence of ahumanized AJvW-2, SEQ ID NO:3.

[0014]FIG. 2(b) is the light chain variable region sequence of ahumanized AJvW-2, SEQ ID NO:4.

[0015]FIG. 3 is a graph of competitive binding properties of murine andhumanized AjvW-2 antibodies (IgG4 and IgG2m3) to von Willebrand factor

DETAILED DESCRIPTION OF THE INVENTION

[0016] In accordance with the present invention, humanizedimmunoglobulins specifically reactive with human von Willebrand factorare provided. These immunoglobulins, which have binding affinities tovWF of at least about 10⁷ M⁻¹ to 10¹⁰ M⁻¹, and preferably 10⁸ M⁻¹ to10¹⁰ M⁻¹ or stronger, are capable of, e g., inhibiting the binding ofvWF to the GPIb protein in the presence of ristocetin or botrocetin.

[0017] The present invention provides novel anti-thrombotic compositionscontaining humanized immunoglobulins specifically capable of binding tothe vWF of humans, and that inhibit RIPA (ristocetin-induced plateletaggregation), BIPA (botrocetin-induced platelet aggregation), and SIPA(shear stress-induced platelet aggregation) reactions of humanplatelets.

[0018] The immunoglobulins can have two pairs of light chain/heavy chaincomplexes, at least one chain comprising one or more mousecomplementarity determining regions functionally joined to humanframework region segments. For example, mouse complementaritydetermining regions, with or without additional naturally-associatedmouse amino acid residues, can be introduced into human frameworkregions to produce humanized immunoglobulins capable of binding to theantigen at affinity levels stronger than about 10⁷ M⁻¹. These humanizedimmunoglobulins are capable of blocking the binding of the CDR-donatingmouse monoclonal antibody to vWF (i.e., AJvW-2).

[0019] The immunoglobulins, including binding fragments and otherderivatives thereof, of the present invention may be produced readily bya variety of recombinant DNA techniques, with ultimate expression intransfected cells, preferably immortalized eukaryotic cells, such asmyeloma or hybridoma cells. Polynucleotides comprising a first sequencecoding for humanized immunoglobulin framework regions and a secondsequence set coding for the desired immunoglobulin complementaritydetermining regions can be produced synthetically or by combiningappropriate cDNA and genomic DNA segments.

[0020] The humanized immunoglobulins may be used in substantially pureform in thrombolytic therapy, that is, removal of preformedintravascular fibrin occulsions. They are also used for prevention andtreatment of atherosclerosis and restenosis after vascular intervention.The humanized immunoglobulins or their complexes can be prepared in apharmaceutically accepted dosage form, which will vary depending on themode of administration.

[0021] The humanized immunoglobulins have a human framework and one ormore complementarity determining regions (CDR's) from immunoglobulinAJvW-2. However, the CDRs from other antibodies that compete withAJvW-2, block the binding of vWF to the GPIb protein in the presence ofristocetin or botrocetin, and/or bind to the same epitope on vWF asAJvW-2 does may also be used. The present immunoglobulins can beproduced economically in large quantities, and find use, for example, inthe treatment of thrombotic diseases in human patients by a variety oftechniques.

[0022] The basic antibody structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy”(about 50-7 kD) chain. The amino-terminal portion of each chainincludes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

[0023] Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, and definethe antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. (See,Fundamental Immunology, Paul, W., Ed., Chapter 7, pgs. 131-166, RavenPress, N.Y. (1984), which is incorporated herein by reference.)

[0024] The variable regions of each light/heavy chain pair form theantibody binding site. The chains all exhibit the same general structureof relatively conserved framework regions joined by three hypervariableregions, also called Complementarity Determining Regions or CDR's (see,“Sequences of Proteins of Immunological Interest,” Kabat, E., et al.,U.S. Department of Health and Human Services, (1987); and Chothia andLesk, J. Mol. Biol., 196, 901-917 (1987), which are incorporated hereinby reference). The CDR's from the two chains of each pair are aligned bythe framework regions, enabling binding to a specific epitope.

[0025] As used herein, the term “immunoglobulin” refers to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Theimmunoglobulins may exist in a variety of forms besides antibodies;including, for example, Fv, Fab, and F(ab′)₂ as well as bifunctionalhybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105(1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426(1988), which are incorporated herein by reference). (See, Hood et al.,Immunology, Benjamin, N. Y., 2^(nd) ed. (1984), Harlow and Lane,Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988)and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which areincorporated herein by reference.).

[0026] Chimeric antibodies are antibodies whose light and heavy chaingenes have been constructed, typically by genetic engineering, fromimmunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalantibody may be joined to human constant (C) segments, such as γ₁ andγ₃. A typical therapeutic chimeric antibody is thus a hybrid proteinconsisting of the V or antigen-binding domain from a mouse antibody andthe C or effector domain from a human antibody, although other mammalianspecies may be used.

[0027] As used herein, the term “framework region” refers to thoseportions of immunoglobulin light and heavy chain variable regions thatare relatively conserved (i.e., other than the CDR's) among differentimmunoglobulins in a single species, as defined by Kabat, et al., op.cit. As used herein, a “human framework region” is a framework regionthat is substantially identical (about 85% or more) to the frameworkregion of a naturally occurring human antibody.

[0028] As used herein, the term “humanized immunoglobulin” refers to animmunoglobulin comprising a human framework, at least one CDR from anon-human antibody, and in which any constant region present issubstantially identical to a human immunoglobulin constant region, i.e.,at least about 85-90%, preferably at least 95% identical. Hence, allparts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of one or more nativehuman immunoglobulin sequences. For example, a humanized immunoglobulinwould not encompass a chimeric mouse variable region/human constantregion antibody.

[0029] Humanized antibodies have at least three potential advantagesover mouse and in some cases chimeric antibodies for use in humantherapy:

[0030] 1. Because the effector portion is human, it may interact betterwith the other parts of the human immune system (e.g., destroy thetarget cells more efficiently by complement-dependent cytotoxicity (CDC)or antibody-dependent cellular cytotoxicity (ADCC)).

[0031] 2. The human immune system should not recognize the framework orC region of the humanized antibody as foreign, and therefore theantibody response against such an injected antibody should be less thanagainst a totally foreign mouse antibody or a partially foreign chimericantibody.

[0032] 3. Injected mouse antibodies have been reported to have ahalf-life in the human circulation much shorter than the half-life ofnormal antibodies (Shaw, D. et al., J. Immunol, 138, 4534-4538 (1987)).Injected humanized antibodies will presumably have a half-lifeessentially identical to naturally occurring human antibodies, allowingsmaller and less frequent doses to be given.

[0033] The present invention relates to recombinant polynucleotidesencoding the heavy and/or light chain CDR's from immunoglobulins capableof binding vWF in the manner of monoclonal antibody AJvW-2. Thepolynucleotides encoding these regions will typically be joined topolynucleotides encoding appropriate human framework regions. As to thehuman framework region, a framework or variable region amino acidsequence of a CDR-providing non-human immunoglobulin is compared withcorresponding sequences in a human immunoglobulin sequence collection,and a sequence having high homology is selected. Exemplarypolynucleotides, which on expression code for the polypeptide chainscomprising the heavy and light chain CDR's of monoclonal antibody AJvW-2are included in FIGS. 1 and 2. Due to codon degeneracy and non-criticalamino-acid substitutions, other polynucleotide sequences can be readilysubstituted for the sequences in FIGS. 1 and 2, as described below.

[0034] The design of humanized immunoglobulins may be carried out asfollows. When an amino acid falls under one of the following categories,the framework amino acid of a human immunoglobulin to be used (acceptorimmunoglobulin) is replaced by a framework amino acid from aCDR-providing non-human immunoglobulin (donor immunoglobulin):

[0035] (a) the amino acid in the human framework region of the acceptorimmunoglobulin is unusual for human immunoglobulins at that position,whereas the corresponding amino acid in the donor immunoglobulin istypical for human immunoglobulins at that position;

[0036] (b) the position of the amino acid is immediately adjacent to oneof the CDR's; or

[0037] (c) the amino acid is capable of interacting with the CDRs in atertiary structure immunoglobulin model (see, Queen et al., op. cit.,and Co et al., Proc. Natl. Acad. Sci.

[0038] USA 88, 2869 (1991), respectively, both of which are incorporatedherein by reference).

[0039] For a detailed description of the production of humanizedimmunoglobulins see, Queen et al., op. cit., and Co et al., op. cit.

[0040] The polynucleotides will typically further include an expressioncontrol polynucleotide sequence operably linked to the humanizedimmunoglobulin coding sequences, including naturally-associated orheterologous promoter regions. Preferably, the expression controlsequences will be eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells, but controlsequences for prokaryotic hosts may also be used. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and, as desired, the collection and purification of the lightchains, heavy chains, light/heavy chain dimers or intact antibodies,binding fragments or other immunoglobulin forms may follow.

[0041] The nucleic acid sequences of the present invention capable ofultimately expressing the desired humanized antibodies can be formedfrom a variety of different polynucleotides (genomic or cDNA, RNA,synthetic oligonucleotides, etc.) and components (e.g., V, J, D, and Cregions), as well as by a variety of different techniques. Joiningappropriate genomic and synthetic sequences is presently the most commonmethod of production, but cDNA sequences may also be utilized (see,European Patent Publication No. 0239400 and Riechmann, L. et al.,Nature, 332, 323-327 (1988), both of which are incorporated herein byreference.)

[0042] Human constant region DNA sequences can be isolated in accordancewith well known procedures from a variety of human cells, but preferablyimmortalized B-cells (see, Kabat op. cit. and WP 87/02671). The CDR'sfor producing the immunoglobulins of the present invention will besimilarly derived from monoclonal antibodies capable of binding to vFWin the manner of AJvW-2 and produced in any convenient mammalian source,including, mice, rats, rabbits, or other vertebrate capable of producingsuch antibodies, by well known methods. Suitable source cells for thepolynucleotide sequences and host cells for immunoglobulin expressionand secretion can be obtained from a number of sources, such as theAmerican Type Culture Collection (Catalogue of Cell Lines andHybridomas, Fifth edition (1985) Rockville, Md., U.S.A., which isincorporated herein by reference).

[0043] In addition to the humanized immunoglobulins specificallydescribed herein, other “substantially homologous” modifiedimmunoglobulins can be readily designed and manufactured utilizingvarious recombinant DNA techniques well known to those skilled in theart. For example, the framework regions can vary from the nativesequences at the primary structure level by several amino acidsubstitutions, terminal and intermediate additions and deletions, andthe like. Moreover, a variety of different human framework regions maybe used singly or in combination as a basis for the humanizedimmunoglobulins of the present invention. In general, modifications ofthe genes may be readily accomplished by a variety of well-knowntechniques, such as site-directed mutagenesis (see, Gillman and Smith,Gene 8, 81-97 (1979) and Roberts S. et al., Nature 328, 731-734 (1987),both of which are incorporated herein by reference.)

[0044] Alternatively, polypeptide fragments comprising only a portion ofthe primary antibody structure may be produced, which fragments possessone or more immunoglobulin activities (e.g., complement fixationactivity). These polypeptide fragments may be produced by proteolyticcleavage of intact antibodies by methods well known in the art, or byinserting stop codons at the desired locations in the vectors usingsite-directed mutagenesis, such as after CH1 to produce Fab fragments orafter the hinge region to produce F(ab′)₂ fragments. Single chainantibodies may be produced by joining VL and VH with a DNA linker (seeHuston et al., op cit., and Bird et al., op cit.). Also because likemany genes, the immunoglobulin-related genes contain separate functionalregions, each having one or more distinct biological activities, thegenes may be fused to functional regions from other genes to producefusion proteins having novel properties.

[0045] As stated previously, the polynucleotides will be expressed inhosts after the sequences have been operably linked to (i.e., positionedto ensure the functioning of) an expression control sequence. Theseexpression vectors are typically replicable in the host organisms eitheras episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors will contain selection markers, e.g.,tetracycline or neomycin, to permit detection of those cells transformedwith the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362,which is incorporated herein by reference). E. coli is one prokaryotichost useful particularly for cloning the polynucleotides of the presentinvention. Other microbial hosts suitable for use include bacilli, suchas Bacillus subtilus, and other enterobacteriacea, such as Salmonella,Serratia, and various Pseudomonas species. In these prokaryotic hosts,one can also make expression vectors, which will typically containexpression control sequences compatible with the host cell (e.g., anorigin of replication). In addition, any number of a variety ofwell-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters willtypically control expression, optionally with an operator sequence, andhave ribosome binding site sequences and the like, for initiating andcompleting transcription and translation. Other microbes, such as yeast,may also be used for expression. Saccharomyces is a preferred host, withsuitable vectors having expression control sequences, such as promoters,including 3-phosphoglycerate kinase or other glycolytic enzymes, and anorigin of replication, termination sequences and the like as desired.

[0046] In addition to microorganisms, mammalian tissue cell culture mayalso be used to express and produce the polypeptides of the presentinvention (see, Winnacker, From Genes to Clones, VCH Publishers, N.Y.,N.Y. (1987), which is incorporated herein by reference). Eukaryoticcells are actually preferred, because a number of suitable host celllines capable of secreting intact immunoglobulins have been developed inthe art, and include the CHO cell lines, various COS cell lines, HeLacells, preferably myeloma cell lines, etc., or transformed B-cells ofhybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, andenhancer (Queen et al., Immunol. Rev. 89, 46-68 (1986), which isincorporated herein by reference), and necessary processing informationsites, such as ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences. Preferred expressioncontrol sequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, cytomegalovirus and the like.

[0047] The vectors containing the polynucleotide sequences of interest(e.g., the heavy and light chain encoding sequences and expressioncontrol sequences) can be transferred into the host cell by well-knownmethods, which vary depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment or electroporation may beused for other cellular hosts. (See, generally, Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1982),which is incorporated herein by reference.)

[0048] Once expressed, the whole antibodies, their dimers, individuallight and heavy chains, or other immunoglobulin forms of the presentinvention can be purified according to standard procedures in the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like (see, generally,Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982), which isincorporated herein by reference). Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses. Once purified,partially or to homogeneity as desired, the polypeptides may then beused therapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings, and the like.(See, generally, Immunological Methods, Vols. I and II, Lefkovits andPernis, eds., Academic Press, New York, N.Y. (1979 and 1981).

[0049] The immunoglobulins of the present invention will typically finduse individually in treating thrombotic diseases in human patients. Thehumanized immunoglobulins and pharmaceutical compositions thereof ofthis invention are particularly useful for parenteral administration,i.e., subcutaneously, intramuscularly, intravenously or intraocularly.The compositions for parenteral administration will commonly comprise asolution of the immunoglobulin or a cocktail thereof dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine, 5% glucose, human albumin solution and the like. Thesesolutions are sterile and generally free of particulate matter. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, tonicity agents, toxicityadjusting agents and the like, for example sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate, sodiumcitrate, etc. The concentration of immunoglobulin in these formulationscan vary widely, i.e., from the less than about 0.5%, usually at leastabout 1% to as much a 15 or 20% by weight and will be selected primarilybased on fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected.

[0050] Thus, a typical pharmaceutical composition for injection could bemade up to contain 1 ml sterile buffered water, and 1-100 mg ofimmunoglobulin. A typical composition for intravenous infusion could bemade up to contain 250 ml of sterile Ringer's solution, and 150 mg ofimmuunoglobulin. Actual methods for preparing parentally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in, for example, Remington's PharmaceuticalScience, 15^(th) ed., Mack Publishing Company, Easton, Pa. (1980), whichis incorporated herein by reference.

[0051] The immunoglobulins of this invention can be frozen orlyophilized for storage and reconstituted in a suitable carrier prior touse. This technique has been shown to be effective with conventionalimmunoglobulins and art-known lyophilization and reconstitutiontechniques can be employed. It will be appreciated by those skilled inthe art that lyophilization and reconstitution can lead to varyingdegrees of immunoglobulin activity loss (e.g., with conventionalimmunoglobulins, IgM antibodies tend to have greater activity loss thanIgG antibodies) and that use levels may have to be adjusted tocompensate.

[0052] The compositions containing the present humanized immunoglobulinsor a cocktail thereof can be administered for therapeutic orprophylactic treatments. In therapeutic application, compositions areadministered to a patient already suffering from thrombotic disease inan amount sufficient to cure or at least partially arrest the diseaseand its complications without causing hemorrhage. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's own immune system, butgenerally range from about 0.1 to 200 mg/kg of immunoglobulin perpatient dose being commonly used. Specific dosing regimens with doses of1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, etc. administered daily, 2 or 3per week, weekly, biweekly, monthly, etc. are all possible and would beselected by a skilled physician depending on the severity of the diseaseand other factors.

[0053] It must be kept in mind that the materials of this invention maygenerally be employed in serious disease states, that is,life-threatening or potentially life-threatening situations. In suchcases, in view of the minimization of extraneous substances and thelower probability of “foreign substance” rejections which are achievedby the present humanized immunoglobulins of this invention, it ispossible and may be felt desirable by the treating physician toadminister substantial excesses of these immunoglobulins.

[0054] Single or multiple administrations of the compositions can becarried out with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the immunoglobulin(s) of this invention sufficient toeffectively treat the patient.

[0055] In particular embodiments, compositions comprising humanizedimmunoglobulins of the present invention may be used to detect vWF.Thus, a humanized immunoglobulin that binds to the antigen determinantidentified by the AJvW-2 antibody may be labeled and used to identifyanatomic sites that contain significant concentrations of vWF. Forexample but not for limitation, one or more labeling moieties may beattached to the humanized immunoglobulin. Exemplary labeling moietiesinclude, but are not limited to, radiopaque dyes, radiocontrast agents,fluorescent molecules, spin-labeled molecules, enzymes, or otherlabeling moieties of diagnostic value, particularly in radiologic ormagnetic resonance imaging techniques.

[0056] Humanized immunoglobulins of the present invention can furtherfind a wide variety of uses in vitro. By way of example, theimmunoglobulins can be used for detection of vWF.

[0057] For diagnostic purposes, the immunoglobulins may either belabeled or unlabeled. Unlabeled immunoglobulins can be used incombination with other labeled antibodies (second antibodies) that arereactive with the humanized immunoglobulin, such as antibodies specificfor human immunoglobulin constant regions. Alternatively, theimmunoglobulins can be directly labeled. A wide variety of labels may beemployed, such as radionuclides, fluors, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors, ligands (particularly haptens),etc. Numerous types of immunoassays are available and are well known tothose skilled in the art.

[0058] Kits can also be supplied for use with the subjectimmunoglobulins in the protection against or detection of a cellularactivity or for the presence of a selected antigen. Thus, the subjectimmunoglobulin composition of the present invention may be provided,usually in a lyophilized form in a container, either alone or inconjunction with additional antibodies specific for the desired celltype. The immunoglobulins, which may be conjugated to a label or toxin,or unconjugated, are included in the kits with buffers, such as Tris,phosphate, carbonate, etc., stabilizers, preservatives, biocides, inertproteins, e.g., serum albumin, or the like, and a set of instructionsfor use. Generally, these materials will be present in less than about5% wt. based on the amount of active immunoglobulin, and usually presentin total amount of at least about 0.001% wt., based again on theimmunoglobulin concentration. Frequently, it will be desirable toinclude an inert extender or excipient to dilute the active ingredients,where the excipient may be present in from about 1 to 99% wt. of thetotal composition. Where a second antibody capable of binding to theimmunoglobulin is employed in an assay, this will usually be present ina separate vial. The second antibody is typically conjugated to a labeland formulated in an analogous manner with the immunoglobulinformulations described above.

[0059] The following examples are offered by way of illustration, not bylimitation. It will be understood that although the examples pertain tothe humanized AJvW-2 antibody, producing humanized antibodies with highbinding affinity for the vWF antigen it is also contemplated using CDR'sfrom other monoclonal antibodies that bind to the same epitope of vWF.

EXAMPLES Example 1 Cloning and Sequencing of Mouse AJvW-2 VariableRegion cDNAs

[0060] Mouse AJvW-2 heavy and light chain variable region cDNAs werecloned from mRNA isolated from hybridoma cells using anchored PCR (Co etal., J. Immunol. 148: 1149 (1992)). The 5′ primers that were usedannealed to poly-dG tails added to the cDNA, and the 3′ primers to theconstant regions. The amplified gene fragments were then inserted intothe plasmid pUC18. Nucleotide sequences were determined from severalindependent clones for both V_(L) and V_(H) cDNA. For the heavy chain, asingle, unique sequence was identified, typical of a mouse heavy chainvariable region. For the light chain, two unique sequences, bothhomologous to murine light chain variable region sequences, wereidentified. However, one sequence was not functional because of amissing nucleotide that caused a frame shift at the V-J junction, andwas identified as the non-productive allele. The other sequence wastypical of a functional mouse kappa chain variable region. The variableregion cDNA sequences of the heavy chain and the functional light chainand the translated amino acid sequences are shown in FIG. 1. The mouseV_(K) sequence belongs to Kabat's mouse kappa chain subgroup V. Themouse V_(H) belongs to Kabat's heavy chain subgroup III(B).

Example 2 Design of Humanized AJvW-2 Variable Regions

[0061] To retain the binding affinity of the mouse antibody in thehumanized antibody, the general procedures of Queen et al. were followed(Queen et al. Proc. Natl. Acad. Sci. USA 86: 10029 (1989) and U.S. Pat.Nos. 5,585,089 and 5,693,762). The choice of framework residues can becritical in retaining high binding affinity. In principle, a frameworksequence from any human antibody can serve as the template for CDRgrafting; however, it has been demonstrated that straight CDRreplacement into such a framework can lead to significant loss ofbinding affinity to the antigen (Tempest et al., Biotechnology 9: 266(1992); Shalaby et al., J Exp. Med. 17: 217 (1992)). The more homologousa human antibody is to the original murine antibody, the less likelywill the human framework introduce distortions into the mouse CDRs thatcould reduce affinity. Based on a sequence homology search against theKabat database (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., U.S. Department of Health and Human Services, 1991),the human antibody I3R was chosen as providing good framework homologyto the mouse AJvW-2 antibody. Other highly homologous human antibodychains would also be suitable to provide the humanized antibodyframework, especially kappa light chains from human subgroup I and heavychains from human subgroup III as defined by Kabat.

[0062] The computer programs ABMOD and ENCAD (Zilber et al.,Biochemistry, Vol. 29, 10032 (1990); Levitt et al., J. Mol. Biol. 168:595 (1983)) were used to construct a molecular model of the AJvW-2variable domain, which was used to locate the amino acids in the AJvW-2framework that are close enough to the CDRs to potentially interact withthem. To design the humanized AJvW-2 heavy and light chain variableregions, the CDRs from the mouse AJvW-2 antibody were grafted into theframework regions of the human I3R antibody. At framework positionswhere the computer model suggested significant contact with the CDRs,the amino acids from the mouse antibody were substituted for theoriginal human framework amino acids. For humanized AJvW-2, this wasdone at residues 28, 48, 49 and 67 of the heavy chain and at residues48, 70 and 71 of the light chain. Furthermore, framework residues thatoccurred only rarely at their positions in the database of humanantibodies were replaced by a human consensus amino acid at thosepositions. For humanized AJvW-2 this was done at residues 1, 78 and 118of the heavy chain and at residues 62, 73 and 83 of the light chain.

[0063] The sequences of the humanized AJvW-2 antibody heavy chain andlight chain variable regions are shown in FIG. 2. However, many of thepotential CDR-contact residues are amenable to substitution by otheramino acids and still allow the antibody to retain substantial affinityfor the antigen. The following table lists a number of positions in theframework where alternative amino acids are suitable (LC=light chain,HC=heavy chain). TABLE I Position Humanized AJvW-2 Alternatives LC-48 VI LC-70 Q D LC-71 Y F HC-28 D T HC-48 I V HC-49 G A, S HC-67 K R

[0064] Likewise, many of the framework residues not in contact with theCDRs in the humanized AJvW-2 heavy and light chains can accommodatesubstitutions of amino acids from the corresponding positions of thehuman I3R antibody, from other human antibodies, by human consensusamino acids, from the mouse AJvW-2 antibody, or from other mouseantibodies, without significant loss of the affinity ornon-immunogenicity of the humanized antibody. The following table listsa number of additional positions in the framework where alternativeamino acids may be suitable. TABLE 2 Position Humanized AJvW-2Alternatives LC-62 F I LC-73 L F LC-83 F I HC-1  E Q HC-78 T S  HC-118 TI, S

[0065] Selection of various alternative amino acids may be used toproduce versions of humanized AJvW-2 that have varying combinations ofaffinity, specificity, non-immunogenicity, ease of manufacture, andother desirable properties. Thus, the examples in the above tables areoffered by way of illustration, not of limitation.

Example 3 Construction of Humanized AJvW-2

[0066] Once the humanized variable region amino acid sequences had beendesigned as described above, genes were constructed to encode them,including signal peptides, splice donor signals and appropriaterestriction sites (FIG. 2). The light and heavy chain variable regiongenes were constructed and amplified using eight overlapping syntheticoligonucleotides ranging in length from approximately 65 to 80 bases(see He et al. J. Immunol. 160: 1029 (1998)). The oligos were annealedpairwise and extended with the Klenow fragment of DNA polymerase I,yielding four double-stranded fragments. The resulting fragments weredenatured, annealed, and extended with Klenow, yielding two fragments.These fragments were denatured, annealed pairwise, and extended onceagain, yielding a full-length gene. The resulting product was amplifiedby polymerase chain reaction (PCR) using Taq polymerase, gel-purified,digested with XbaI, gel-purified again, and subcloned into the XbaI siteof the pVk, pVg4 or pVg2.M3 expression vector. The pVk vector for lightchain expression has been previously described (see Co et al., J.Immunol. 148:1149 (1992)). The pVg4 vector for heavy chain expressionwas constructed by replacing the XbaI-BamHI fragment of pVg1 containingthe g1 constant region gene (see Co et al., J. Immunol. 148: 1149(1992)) with an approximately 2000 bp fragment of the human g4 constantregion gene (Ellison and Hood, Proc. Natl. Acad. Sci. USA 79: 1984(1982)) that extended from the HindIII site preceding the C_(H)1 exon ofthe g4 gene to 270 bp after the NsiI site following the C_(H)4 exon ofthe gene. The pVg2.M3 vector for expression of gamma 2 chain has beenpreviously described (see Cole, et al., J. Immunol. 159: 3613 (1997)).The pVg2.M3 is a variant of the human wildtype IgG2 by replacing theamino acids Val and Gly at positions 234 and 237 with Ala. The varianthas a reduced interaction with its Fc receptors and thus has minimalantibody effector activity.

[0067] The structure of the final plasmids were verified by nucleotidesequencing and restriction mapping. All DNA manipulations were performedby standard methods well-known to those skilled in the art.

[0068] Two humanized AJvW-2, an IgG4 and an IgG2.M3, were generated forcomparative studies. To construct a cell line producing humanizedAJvW-2, the respective heavy chain and light chain plasmids weretransfected into the mouse myeloma cell line Sp2/0-Ag14 (ATCC CRL 1581).Before transfection, the heavy and light chain-containing plasmids werelinearized using restriction endonucleases. The kappa chain and thegamma2 heavy chain were linearized using FspI; the gamma 4 chain waslinearized using BstZ17I. Approximately 20 μg of each plasmid wastransfected into 1×107 cells in PBS. Transfection was by electroporationusing a Gene Pulser apparatus (BioRad) at 360 V and 25 μFD capacitanceaccording to the manufacturer's instructions. The cells from eachtransfection were plated in four 96-well tissue culture plates, andafter two days, selection medium (DMEM, 10% FCS, 1×HT supplement(Sigma), 0.25 mg/ml xanthine, 1 μg/ml mycophenolic acid) was applied.

[0069] After approximately two weeks, the clones that appeared werescreened for antibody production by ELISA. Antibody from ahigh-producing clone was prepared by growing the cells to confluency inregular medium (DMEM with 10% FCS), then replacing the medium with aserum-free medium (Hybridoma SMF; Gibco) and culturing until maximumantibody titers were achieved in the culture. The culture supernatantwas run through a protein A-Sepharose column (Pharmacia); antibody waseluted with 0.1 M Glycine, 100 mM NaCl, pH 3, neutralized andsubsequently exchanged into phosphate-buffered saline (PBS). The purityof the antibody was verified by analyzing it on an acrylamide gel, andits concentration was determined by an OD₂₈₀ reading, assuming 1.0 mg ofantibody protein has an OD₂₈₀ reading of 1.4.

Example 4 Properties of Humanized AJvW-2

[0070] The affinity of the murine and humanized AJvW-2 antibodies forvon Willebrand factor (vWF) was determined by competitive binding withbiotinylated murine AJvW-2 antibody. The procedure for the experiment isdescribed below:

[0071] 1. vWF solution was diluted to 8 ug/ml with TBS (20 mM Tris pH7.4+0.15 M NaCl). 50 ul was dispensed to each well of a 96-well NUNCMaxisorp plate (VWR Scientific Product) and incubated overnight at 4° C.

[0072] 2. The plate was washed once with TBS, blocked by adding 200ul/well of a blocking solution (TBS+5% BSA) and incubated for 3 hr atroom temperature.

[0073] 3. The plate was washed three times with TBS.

[0074] 4. Murine AJvW-2 was previously biotinylated usingsulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce, Rockford, Ill.,product number 21335) according to the manufacturer's instruction. Thebiotinylated antibody was diluted to 0.5 ug/ml in TBS+0.1% BSA.

[0075] 5. Eight 4-fold serial dilutions of cold competitor murine andhumanized antibodies were prepared in TBS+0.1% BSA, starting at 25ug/ml.

[0076] 6. The following solutions were added to each well of the vWFcoated plate: 25 ul TBS+0.1% BSA+10% DMSO, 100 ul of cold competitorantibody (murine, humanized IgG2m3 or humanized IgG4) and 25 ul ofbiotinylated antibody, and incubate at room temperature for 1 hr withgentle shaking.

[0077] 7. The plate was washed three times with a washing solution(TBS+0.05% Tween-20) and stained with the ImmunoPure ABC PhosphataseStaining Kits (Pierce, Rockford, Ill.) according to the manufacturer'sinstruction. Specifically, a solution was prepared by adding 2 drops ofreagent A (avidin) and 2 drops of reagent B (biotinylated alkalinephosphatase) to 50 ml of TBS+0.1% BSA. 50 ul of the prepared solutionwas added to each well of the 96-well plate and incubated at roomtemperature for 1 hr.

[0078] 8. The plate was washed three times with the washing solution anddeveloped with Alkaline Phosphatase substrate (Sigma, St. Louis, Mo.).

[0079] 9. Absorbance was measured at 405 nm and plotted against theconcentration of competitor antibodies.

[0080] The result, shown in FIG. 3, demonstrated that the humanizedAJvW-2 IgG4 and IgG2m3 compete equally well with the biotinylated murineantibody when compared to the unlabeled murine antibody, suggesting thatthe two humanized antibodies have similar binding affinities and thereis no significant difference in the affinity of the humanized antibodiesand the murine antibody to the antigen.

[0081]FIG. 1 shows the cDNA and translated amino acid sequences of theheavy chain (A) and light chain (B) variable regions of the murineAJvW-2 antibody. The complementarity determining regions (CDRs) areunderlined and the first amino acids of the mature chains are doubleunderlined.

[0082]FIG. 2 shows the DNA and translated amino acid sequences of theheavy chain (A) and light chain (B) variable regions of the humanizedAJvW-2 antibody. The complementarity determining regions (CDRS) areunderlined and the first amino acids of the mature chains are doubleunderlined.

[0083]FIG. 3 is a graph of competitive binding properties of murine andhumanized AJvW-2 antibodies (IgG4 and IgG2m3) to von Willebrand factor.Increasing concentrations of cold competitor antibody were incubatedwith von Willebrand factor in the presence of biotinylated tracer murineAJvW-2. Absorbance was measured and plotted against the concentration ofthe unlabeled competitor antibodies.

[0084] Obviously numerous variations of the invention are possible inlight of the above teachings. Therefore, within the scope of theappended claims, the invention may be practiced otherwise than asspecifically described.

1 8 1 417 DNA Mus musculus CDS (1)..(417) 1 atg gat ttt ggg ctg att tttttt att gtt gct ctt tta aaa ggg gtc 48 Met Asp Phe Gly Leu Ile Phe PheIle Val Ala Leu Leu Lys Gly Val 1 5 10 15 cag tgt gag gtg aaa ctt ctcgag tct gga ggt ggc ctg gtg cag act 96 Gln Cys Glu Val Lys Leu Leu GluSer Gly Gly Gly Leu Val Gln Thr 20 25 30 gga gga tcc ctg aaa ctc tcc tgtgca gcc tca gga ttc gat ttt agt 144 Gly Gly Ser Leu Lys Leu Ser Cys AlaAla Ser Gly Phe Asp Phe Ser 35 40 45 aga ttc tgg atg agt tgg gtc cgg caggct cca ggg aaa ggg cta gaa 192 Arg Phe Trp Met Ser Trp Val Arg Gln AlaPro Gly Lys Gly Leu Glu 50 55 60 tgg att gga gaa gtt aat cca gat aac aatacg atg aac tat acg cca 240 Trp Ile Gly Glu Val Asn Pro Asp Asn Asn ThrMet Asn Tyr Thr Pro 65 70 75 80 tct cta aag gat aaa ttc atc atc tcc agagac aac gcc aaa aat acg 288 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg AspAsn Ala Lys Asn Thr 85 90 95 ctg tac ctg caa atg agt caa gtg aga tct gaggac aca gcc ctt tac 336 Leu Tyr Leu Gln Met Ser Gln Val Arg Ser Glu AspThr Ala Leu Tyr 100 105 110 tac tgt gca aga cct ccc tac tat ggt agc tacggg ggg ttt gct tac 384 Tyr Cys Ala Arg Pro Pro Tyr Tyr Gly Ser Tyr GlyGly Phe Ala Tyr 115 120 125 tgg ggc caa ggg act ctg gtc tct gtc tcg cca417 Trp Gly Gln Gly Thr Leu Val Ser Val Ser Pro 130 135 2 139 PRT Musmusculus 2 Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys GlyVal 1 5 10 15 Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu ValGln Thr 20 25 30 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe AspPhe Ser 35 40 45 Arg Phe Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys GlyLeu Glu 50 55 60 Trp Ile Gly Glu Val Asn Pro Asp Asn Asn Thr Met Asn TyrThr Pro 65 70 75 80 Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn AlaLys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Ser Gln Val Arg Ser Glu Asp ThrAla Leu Tyr 100 105 110 Tyr Cys Ala Arg Pro Pro Tyr Tyr Gly Ser Tyr GlyGly Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Ser Val Ser Pro130 135 3 381 DNA Mus musculus CDS (1)..(381) 3 atg agt gtg ccc act caggtc ctg ggg ttg ctg ctg ctg tgg ctt aca 48 Met Ser Val Pro Thr Gln ValLeu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 gat gcc aga tgt gac atccag atg act cag tct cca gcc tcc cta tct 96 Asp Ala Arg Cys Asp Ile GlnMet Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30 gta tct gtg gga gaa act gtcacc atc aca tgt cga gca agt gag aat 144 Val Ser Val Gly Glu Thr Val ThrIle Thr Cys Arg Ala Ser Glu Asn 35 40 45 att tac aat aat tta gct tgg tatcag cag aga cag gga aaa tct cct 192 Ile Tyr Asn Asn Leu Ala Trp Tyr GlnGln Arg Gln Gly Lys Ser Pro 50 55 60 cag ctc ctg gtc tat gct gca aca aactta gca gat ggt gtg cca tca 240 Gln Leu Leu Val Tyr Ala Ala Thr Asn LeuAla Asp Gly Val Pro Ser 65 70 75 80 agg ttc agt ggc agt gga tca ggc acacag tat tcc ctc aag atc gac 288 Arg Phe Ser Gly Ser Gly Ser Gly Thr GlnTyr Ser Leu Lys Ile Asp 85 90 95 agc ctg cag tct gaa gat ttt ggg agt tattac tgt caa cat ttg tgg 336 Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr TyrCys Gln His Leu Trp 100 105 110 act tct ccg tac acg ttc gga ggg ggg accaag ctg gaa ata aaa 381 Thr Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys LeuGlu Ile Lys 115 120 125 4 127 PRT Mus musculus 4 Met Ser Val Pro Thr GlnVal Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys AspIle Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30 Val Ser Val Gly GluThr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn 35 40 45 Ile Tyr Asn Asn LeuAla Trp Tyr Gln Gln Arg Gln Gly Lys Ser Pro 50 55 60 Gln Leu Leu Val TyrAla Ala Thr Asn Leu Ala Asp Gly Val Pro Ser 65 70 75 80 Arg Phe Ser GlySer Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asp 85 90 95 Ser Leu Gln SerGlu Asp Phe Gly Ser Tyr Tyr Cys Gln His Leu Trp 100 105 110 Thr Ser ProTyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 5 417 DNAArtificial Sequence CDS (1)..(417) Description of ArtificialSequenceSYNTHETIC DNA 5 atg gat ttt ggg ctg att ttt ttt att gtt gct ctttta aaa ggg gtc 48 Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu LeuLys Gly Val 1 5 10 15 cag tgt gag gtg caa ctt gtc gag tct gga ggt ggacta gtg cag cct 96 Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Gln Pro 20 25 30 gga gga tca ctg aga ctc tcc tgt gca gcc tca gga ttcgat ttt agt 144 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe AspPhe Ser 35 40 45 aga ttc tgg atg agt tgg gtc cgg cag gct cca ggg aaa gggctc gag 192 Arg Phe Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu 50 55 60 tgg att gga gaa gtt aat cca gat aac aat acg atg aac tat acgcca 240 Trp Ile Gly Glu Val Asn Pro Asp Asn Asn Thr Met Asn Tyr Thr Pro65 70 75 80 tct cta aag gat aaa ttc acc atc tcc aga gac aac gcc aaa aatacg 288 Ser Leu Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr85 90 95 ctg tac ctg caa atg aac tca ttg aga gct gag gac acg gcc gtt tac336 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100105 110 tac tgt gca aga cct ccc tac tat ggt agc tac ggg ggg ttt gct tac384 Tyr Cys Ala Arg Pro Pro Tyr Tyr Gly Ser Tyr Gly Gly Phe Ala Tyr 115120 125 tgg ggc caa ggg act ctg gtc acc gtc tcc tca 417 Trp Gly Gln GlyThr Leu Val Thr Val Ser Ser 130 135 6 139 PRT Artificial SequenceDescription of Artificial SequenceSYNTHETIC DNA 6 Met Asp Phe Gly LeuIle Phe Phe Ile Val Ala Leu Leu Lys Gly Val 1 5 10 15 Gln Cys Glu ValGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly Gly Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 35 40 45 Arg Phe Trp MetSer Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly GluVal Asn Pro Asp Asn Asn Thr Met Asn Tyr Thr Pro 65 70 75 80 Ser Leu LysAsp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100 105 110 Tyr CysAla Arg Pro Pro Tyr Tyr Gly Ser Tyr Gly Gly Phe Ala Tyr 115 120 125 TrpGly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 7 381 DNA ArtificialSequence CDS (1)..(381) Description of Artificial SequenceSYNTHETIC DNA7 atg agt gtg ccc act cag gtc ctg ggg ttg ctg ctg ctg tgg ctt aca 48 MetSer Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15gat gcc aga tgt gac atc cag atg act cag tct cca tcc tcc cta tct 96 AspAla Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 gcatct gtg gga gac agg gtc acc atc aca tgt cga gca agt gag aat 144 Ala SerVal Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn 35 40 45 att tacaat aat tta gct tgg tat cag cag aaa ccg gga aaa gct cct 192 Ile Tyr AsnAsn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 aag cta ctagtc tat gct gca aca aac tta gca gat ggt gtg cca tca 240 Lys Leu Leu ValTyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser 65 70 75 80 agg ttc agtggc agt gga tca ggc aca cag tat acc ctc acg atc agc 288 Arg Phe Ser GlySer Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser 85 90 95 agc ctc cag cctgag gat ttt gcg act tat tac tgt caa cat ttg tgg 336 Ser Leu Gln Pro GluAsp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp 100 105 110 act tct ccg tacacg ttc gga ggg ggg acc aag gtg gaa ata aaa 381 Thr Ser Pro Tyr Thr PheGly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 8 127 PRT ArtificialSequence Description of Artificial SequenceSYNTHETIC DNA 8 Met Ser ValPro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp AlaArg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala SerVal Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn 35 40 45 Ile TyrAsn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys LeuLeu Val Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser 65 70 75 80 ArgPhe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser 85 90 95 SerLeu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp 100 105 110Thr Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125

We claim:
 1. A humanized immunoglobulin which binds to human von Willebrand factor.
 2. The immunoglobulin of claim 1 which competes with mouse antibody AJvW-2 for specific binding to von Willebrand factor.
 3. The immunoglobulin of claim 1 which is an antibody comprising a heavy chain variable region shown in FIG. 2a (SEQ. ID. NO. 3) and a light chain variable region shown in FIG. 2b (SEQ. ID. NO. 4).
 4. A humanized immunoglobulin that is a humanized form of mouse antibody AJvW-2.
 5. The humanized immunoglobulin of claim 1, comprising: complementarity determining regions from the mouse AJvW-2 antibody, and heavy and light chain variable region frameworks from human I3R antibody heavy and light chain frameworks, provided that at least one position selected from the group consisting of LC-48, LC-70, LC-71, HC-28, HC-48, HC-49 and HC-67 is occupied by the amino acid present in the equivalent position of the mouse AJvW-2 antibody heavy or light chain variable region framework, which humanized antibody specifically binds to vWF with an affinity constant between 10⁷ M⁻¹ and ten-fold the affinity of the mouse AJvW-2 antibody.
 6. The humanized immunoglobulin of claim 5, which is an antibody wherein each position selected from the group consisting of LC-48, LC-70, LC-71, HC-28, HC-48, HC-49 and HC-67 is occupied by the amino acid present in the equivalent position of the mouse AJvW-2 antibody heavy or light chain variable region framework.
 7. The humanized antibody of claim 6, wherein at least one position selected from the LC-62, LC-73, LC-83, HC-1, HC-78 and HC-118 is occupied by an amino acid present in the equivalent position of a human antibody heavy or light chain consensus sequence.
 8. The humanized immunoglobulin of claim 5 comprising a heavy chain variable region shown in FIG. 2a (SEQ. ID. NO. 3) and a light chain variable region shown in FIG. 2b (SEQ. ID. NO. 4), wherein one or more amino acid positions may be substituted by alternatives as shown in Tables 1 and
 2. 9. The humanized immunoglobulin of claim 1, comprising a humanized heavy chain having at least 85% identity with the humanized heavy chain shown in FIG. 2a (SEQ. ID. NO. 3) and a humanized light chain having at least 85% sequence identity with the humanized light chain showing in FIG. 2b (SEQ. ID. NO. 4), provided that at least one position selected from the group consisting of LC-48, LC-70, LC-71, HC-28, HC-48, HC-49 and HC-67 is occupied by the amino acid present in the equivalent position of the mouse AJvW-2 antibody heavy or light chain variable region framework.
 10. The immunoglobulin of claim 1, comprising two pairs of light/heavy chain dimers, wherein each chain comprises a variable region and a constant region.
 11. The immunoglobulin of claim 1, which is a Fab fragment or a F(ab′)_(2.)
 12. The humanized immunoglobulin of claim 1 having complementarity determining regions (CDRs) from AJvW-2 and heavy and light chain variable region frameworks wherein the sequence of the heavy chain variable region framework is a consensus sequence of human immunoglobulin heavy chain variable region frameworks.
 13. The humanized immunoglobulin of claim 1, which has an IgG₂ or IgG4 immunoglobulin subtype.
 14. The humanized immunoglobulin of claim 1, wherein the constant region is a Cγ2 Cγ4 region.
 15. A method of producing an immunoglobulin comprising: culturing a cell line that encodes heavy and light chain chains of a humanized immunoglobulin, whereby a humanized antibody which competes with mouse antibody AJvW-2 is expressed; and recovering said humanized antibody.
 16. The method of claim 15, further comprising formulating the humanized antibody with a pharmaceutically acceptable carrier to produce a pharmaceutical composition.
 17. A pharmaceutical composition comprising: a humanized immunoglobulin which competes with mouse antibody AJvW-2 for specific binding to von Willebrand factor, and a pharmaceutically acceptable carrier.
 18. A method of treating a patient having or at risk of a thrombotic disease or athelosclerosis, comprising: administering to said patient an effective dose of a humanized immunoglobulin which competes with mouse antibody AJvW-2 for specific binding to von Willebrand factor.
 19. The method of claim 18, wherein the immunoglobulin is a humanized form of mouse antibody AJvW-2.
 20. The method of claim 18, wherein the immunoglobulin comprises a heavy chain variable region shown in FIG. 2a (SEQ. ID. NO. 3) and a light chain variable region shown in FIG. 2b (SEQ. ID. NO. 4).
 21. A cell line that produces a human immunoglobulin which competes with mouse antibody AJvW-2 for specific binding to von Willebrand factor. 